Design, synthesis and anti-mycobacterial evaluation of some new N-phenylpyrazine-2-carboxamides

Article abstract of DOI:10.1515/chempap-2015-0246

N-Phenylpyrazine-2-carboxamides (anilides of pyrazinoic acids with simple substituents in various positions) were previously shown to possess significant biological activities in vitro, markedly anti-mycobacterial and photosynthesis-inhibiting activity. Based on structure-activity relationships (SAR) extracted from previously published series, 25 new anilides of non-substituted pyrazinoic acid (POA), 5-CH₃-POA, 6-Cl-POA, 5-tert-butyl-POA and 5-tert-butyl-6-Cl-POA were designed and synthesised. The phenyl part was substituted with simple hydrophobic substituents chosen from methyl and halogens. 5-tert-Butyl-N-(5-fluoro-2-methylphenyl)pyrazine-2-carboxamide (9), N-(3-chloro-4-methylphenyl)-5-methylpyrazine-2-carboxamide (12), 6-chloro-N-(3-chloro-4-methylphenyl)pyrazine-2-carboxamide (13) and 6-chloro-N-(5-iodo-2-methylphenyl)pyrazine-2-carboxamide (18) possessed whole cell anti-mycobacterial activity in vitro against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) of around 10 μM. Importantly, no cytotoxicity in the HepG2 model was detected in vitro at the concentrations tested and the estimated IC₅₀ values were in hundreds of μM, indicating promising selectivity. N-(3-Chloro-4-methylphenyl)pyrazine-2-carboxamide (11) and N-(4-chloro-2-iodophenyl)pyrazine-2-carboxamide (21) exerted significant activity against Mycobacterium kansasii with MIC 12.6 μM and 8.7 μM, respectively. No activity was detected against Mycobacterium avium. SAR were in accordance with those observed for the derivatives previously published.

Full text of DOI:10.1515/chempap-2015-0246

Chemical Papers
DOI: 10.1515/chempap-2015-0246
ORIGINAL PAPER
Design, synthesis and anti-mycobacterial evaluation of some new
-phenylpyrazine-2-carboxamides
a
aJan Zitko*, Barbora Servusová-Vaňásková*, a,bPavla Paterová,
a
a
a
aLucie Navrátilová, František Trejtnar, Jiří Kuneš, Martin Doležal
aFaculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové 50005, Czech Republic
bDepartment of Clinical Microbiology, University Hospital, Hradec Králové 50005, Czech Republic
Received 26 August 2015; Revised 30 September 2015; Accepted 21 October 2015
N-Phenylpyrazine-2-carboxamides (anilides of pyrazinoic acids with simple substituents in vari-
ous positions) were previously shown to possess significant biological activities in vitro, markedly
anti-mycobacterial and photosynthesis-inhibiting activity. Based on structure-activity relation-
ships (SAR) extracted from previously published series, 25 new anilides of non-substituted
pyrazinoic acid (POA), 5-CH3-POA, 6-Cl-POA, 5-tert-butyl-POA and 5-tert-butyl-6-Cl-POA
were designed and synthesised. The phenyl part was substituted with simple hydrophobic sub-
stituents chosen from methyl and halogens. 5-tert-Butyl-N-(5-fluoro-2-methylphenyl)pyrazine-2-
carboxamide (9), N-(3-chloro-4-methylphenyl)-5-methylpyrazine-2-carboxamide (12), 6-chloro-N-
(3-chloro-4-methylphenyl)pyrazine-2-carboxamide (13) and 6-chloro-N-(5-iodo-2-methylphenyl)py-
razine-2-carboxamide (18) possessed whole cell anti-mycobacterial activity in vitro against My-
cobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) of around 10 µM.
Importantly, no cytotoxicity in the HepG2 model was detected in vitro at the concentrations
tested and the estimated IC50 values were in hundreds of µM, indicating promising selectivity.
N-(3-Chloro-4-methylphenyl)pyrazine-2-carboxamide (11) and N-(4-chloro-2-iodophenyl)pyrazine-
2-carboxamide (21) exerted significant activity against Mycobacterium kansasii with MIC 12.6 µM
and 8.7 µM, respectively. No activity was detected against Mycobacterium avium. SARs were in
accordance with those observed for the derivatives previously published.
c
ꢀ 2015 Institute of Chemistry, Slovak Academy of Sciences
Keywords: anilide, anti-mycobacterial activity, cytotoxicity in vitro, lipophilicity, pyrazinoic acid
Introduction
approximately 13 % of new TB cases in 2013) consti-
tutes a serious problem and emphasises the need for
novel anti-tubercular agents. Accordingly, the search
for new anti-tuberculosis drugs is an important re-
search topic (Nemeček et al., 2013; Krátký et al.,
2015).
Pyrazinamide (PZA), a first-line anti-tubercular
agent, is a model compound used as a starting point
for the design of N-phenylpyrazine-2-carboxamides
presented in this paper. Although PZA has been used
in clinical practice since the 1950s, its complex mech-
anism of action is not yet fully understood. The non-
Tuberculosis (TB) is one of the most lethal
and frequent infectious diseases worldwide. Accord-
ing to the World Health Organisation (2014), there
were 9 million new cases of TB and 1.5 million
deaths associated with TB (including 0.36 million
deaths of HIV-positives) in 2013. The alarming in-
crease in drug-resistant TB strains, namely multi-
drug-resistant (MDR) and extensively drug-resistant
(XDR), as well as the increasing number of patients
co-infected with HIV (1.1 million, which represents
*Corresponding author, e-mail: jan.zitko@faf.cuni.cz, barbora.servusova@faf.cuni.cz
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specific mechanism based on the accumulation of the
pyrazinoic acid (POA; active metabolite) in the my-
cobacterial cell and the acidification of the cytoplasm
is generally recognised. However, recent studies have
shown that POA also has a specific intracellular tar-
get. Most importantly, Shi et al. (2011) showed that
POA prevents the binding of tmRNA to the riboso-
mal protein S1 (RpsA), leading to the inhibition of
trans-translation, the vital process of rescuing stalled
ribosomes. Recently, Yang et al. (2015) determined the
structure of POA-RpsA co-crystallised complex and,
on the basis of the interactions observed, proposed
possible favourable modifications of POA in respect of
anti-mycobacterial activity. This clearly demonstrates
that PZA, POA and their derivatives are still in focus.
For several years, N-phenylpyrazine-2-carbox-
amides (i.e. anilides of POA) have been the focus
of the working group led by Doležal at the Fac-
ulty of Pharmacy in Hradec Králové (Hradec Králové,
Czech Republic). More than one hundred anilides were
synthesised and evaluated as potential anti-infective
agents with anti-mycobacterial, antibacterial or an-
tifungal activity (Dolezal et al., 2002, 2006, 2008,
2009, 2010). Many N-phenylpyrazine-2-carboxamides
also proved to be effective photosynthesis inhibitors
in the spinach chloroplast model, interrupting the
photosynthetic electron transport (PET) in photo-
system II (PS II) (Dolezal et al., 2002, 2006, 2008,
2010). For further information and SAR on the PET-
inhibiting activity, (Dolezal & Kralova, 2011). Sev-
eral other research groups have focused on potential
anti-tuberculosis agents containing the carboxanilide
pattern. Gonec et al. (2015) prepared and tested
derivatives of naphthalene-2-carboxanilides (Kos et
al., 2015a), and quinoline-2-carboxanilides (Kos et
al., 2015b) as potential anti-tuberculosis agents. The
working group led by Vinsova focused on anti-
tuberculosis salicylanilides (Kratky & Vinsova, 2011).
Recently, these researchers developed salicylanilides
esterified with POA (compounds combining two ac-
tive anti-tuberculosis fragments – the salicylanilide
and POA). These compounds exhibited promising mi-
cromolar to sub-micromolar activity in vitro against
multidrug-resistant mycobacterial strains (Kratky et
al., 2014).
Fig. 1. General formula of N-phenylpyrazine-2-carboxamides;
X – halogen.
ordinating Facility (TAACF), established by the Na-
tional Institute of Allergy and Infectious Diseases (NI-
AID; Bethesda, MD, USA). In the primary screening
against Mycobacterium tuberculosis H37Rv (ATCC
27294), the activity was expressed as a percentage of
growth inhibition at 6.25 g mL−1 in the BACTEC
12B medium using the microplate alamar blue assay
(MABA) (Collins & Franzblau, 1997).
These comprehensive data were used to extract
the basic structure-activity relationships (SAR) in
respect of the calculated lipophilicity of the com-
pounds and their substitution in the anilide part
of the molecule (R3; Fig. 1). Analysis showed that
the most anti-mycobacterial activity-enhancing sub-
stituents R3 were 3-CF3, 4-CH3, 4-CH(CH3)2, 3-F,
and 3,5-CF3 (Dolezal et al., 2012), that is, the
electron-withdrawing substituent in the meta posi-
tion and the electron-donating substituent in the
para position. In the current study, the two counter-
acting substituents were combined in one molecule
and the R3 = 3-Cl-4-CH3 pattern was proposed
as the substitution of choice. This was also in-
spired by the fact that a previous publication showed
N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide to
be one of the most active anilides examined, inhibit-
ing the growth of M. tuberculosis H37Rv by 95 %
at 6.25 g mL−1 (Dolezal et al., 2011). Other sub-
stituent patterns R3 of the compounds in this article
(Fig. 2) originated on the basis of the mutual exchange
of various halogen atoms (chlorine for other halogens)
and/or positional isomerism, to further confirm that
the 3,4-disubstitution is superior to other combina-
tions.
The most comprehensive review (Dolezal et al.,
2012) published on the anti-mycobacterial activity
of the N-phenylpyrazine-2-carboxamides in question
contains the activity data on 103 derivatives of general
formula depicted in Fig. 1. The anilides reported were
derived from non-substituted POA, 6-Cl-POA, 5-tert-
butyl-POA and 5-tert-butyl-6-Cl-POA. The phenyl
ring was mono-, di- or tri-substituted with small sub-
stituents R3 (Fig. 1) chosen from short alkyl (methyl,
isopropyl), methoxy, halogen, CF3 and hydroxy sub-
stituents. The anti-mycobacterial activity was mea-
sured in a high-throughput screening campaign run by
the Tuberculosis Antimicrobial Acquisition and Co-
From previous experience with different series
of pyrazine derivatives evaluated as potential anti-
tuberculosis agents, it is known that activity usually
grows with increasing lipophilicity to a certain point
but a further increase in lipophilicity often leads to di-
minished or lost activity of such derivatives. For exam-
ple, this pertained for various series of PZA derivatives
with alkylamino substituents with increasing lengths
of carbon chain, where the activity culminated in
hexyl- or heptyl-amino derivatives (Servusova et al.,
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iii
Fig. 2. Design of title compounds (1–25) based on previously determined SAR.
2014; Servusova-Vanaskova et al., 2015a, 2015b; Zitko
et al., 2015). Increased lipophilicity of a compound is
beneficial for permeation of the highly lipophilic my-
cobacterial envelope (Brennan, 2003). On the other
hand, excessive lipophilicity is associated with insuf-
ficient water solubility and the ability of a compound
to reach therapeutic concentrations is impaired. These
two counteracting phenomena give rise to the opti-
mal range of lipophilicity. The MycPermCheck model
developed by Merget et al. (2013) is based on com-
pounds with whole cell anti-mycobacterial activity in
vitro found in the literature. The model establishes
the optimal lipophilicity range for good permeation as
QPlogPo/w = 2.779–4.479 where QPlogPo/w is the
octanol/water partition coefficient predicted by the
QikProp module (Schro¨dinger, USA) (Merget et al.,
2013). The anilides designed in this study exhibited in-
creased lipophilicity due to their R3 substituent, hence
they were used to probe the upper lipophilicity limit
for the anti-mycobacterial activity in vitro.
purified using an automated chromatograph Combi-
Flash Rf (Teledyne Isco, USA) using columns filled
with Kieselgel 60, 0.040–0.063 mm (Merck); gradient
elution (hexane/ethyl acetate), detection wavelength
of 260 nm, monitor wavelength of 280 nm. NMR anal-
ysis was performed on a Varian Mercury VX-BB 300
(Varian, USA) at 300 MHz for 1H and 75 MHz for
13C or on a Varian Mercury-Vx BB 500 (Varian) at
500 MHz for 1H and 125 MHz for 13C. The chemi-
cal shifts were recorded in δ and were indirectly ref-
erenced to tetramethylsilane (TMS). The IR spectra
were measured in ATR mode using a Ge crystal-plate
on a Nicolet Impact 400 (Nicolet, USA). Elementary
analysis was performed on a CE Instruments EA-1110
CHN analyser (CE Instruments, UK). Melting points
were determined on a Stuart SMP30 melting point
apparatus (Bibby Scientific Limited, UK) and are un-
corrected. LogP (the logarithm of the partition co-
efficient for n-octanol/water) values were calculated
using the CS ChemBioDraw Ultra program ver. 14.0
(CambridgeSoft, USA).
6-Chloropyrazine-2-carboxylic (Abe et al., 1969)
(6-Cl-POA), 5-tert-butylpyrazine-2-carboxylic (Dole-
zal et al., 1999) (5-tert-Bu-POA) and 5-tert-butyl-
6-chloropyrazine-2-carboxylic acid (Dolezal et al.,
1999) (5-tert-Bu-6-Cl-POA) were synthesised follow-
ing the known synthetic procedures described else-
where (Dolezal et al., 1999; Servusova et al., 2014;
Venturello & D’Aloisio, 1986). The analytical data
of the prepared starting acids were fully in accor-
dance with the data in the literature and 1H NMR
as well as the melting points are available in the
Supplementary Data. POA and 5-methylpyrazine-2-
In summary, 25 new anilides, which had not previ-
ously been described, were designed, synthesised and
tested in vitro for anti-mycobacterial activity against
M. tuberculosis H37Rv and non-tuberculous mycobac-
terial strains of M. kansasii and M. avium.
Experimental
All the organic solvents used for the synthesis were
of analytical grade. Unless otherwise stated, all chem-
icals were purchased from Sigma–Aldrich (Germany).
The reactions were monitored using Merck Silica 60
F254 TLC plates (Merck, Germany). Compounds were
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J. Zitko et al./Chemical Papers
carboxylic acid (5-CH3-POA) were purchased from
Sigma–Aldrich and used as received.
Cytotoxicity measurement
The human hepatocellular liver carcinoma cell line
HepG2 (passage 40–41) purchased from Health Pro-
tection Agency Culture Collections (ECACC, UK)
was routinely cultured in a minimum essentials eagle
medium (MEM; Sigma–Aldrich) supplemented with
10 vol. % foetal bovine serum (PAA, Austria), 1 %
L-glutamine solution (Sigma–Aldrich) and 1 vol. %
non-essential amino acid solution (Sigma–Aldrich) in
a humidified atmosphere containing 5 % of CO2 at
37◦C. For sub-culturing, the cells were harvested after
trypsin/EDTA (Sigma–Aldrich) treatment at 37◦C.
The cells treated with the tested substances were used
as the experimental groups and untreated HepG2 cells
were used as the control groups. The cells were seeded
in a density of 1 × 104 cells per well in a 96-well
plate. The next day they were treated with the tested
substances dissolved in DMSO (maximal incubation
concentration of DMSO was 1 vol. %). The tested
compounds were prepared at incubation concentra-
tions of 1–7500 M. The treatment was carried out
in triplicates in a humidified atmosphere containing
5 % of CO2 at 37◦C for 24 h. The controls repre-
senting 100 % cell viability, 0 % cell viability (cells
treated with 10 vol. % DMSO), no-cell controls and
vehiculum controls were incubated in triplicate simul-
taneously. After 24 h exposure, the reagent from the
kit CellTiter 96ꢀ Aqueous one solution cell prolifer-
ation assay (Promega, USA) was added according to
the manufacturer’s recommendation. After 2 h incuba-
tion at 37◦C in a humidified, 5 % of CO2 atmosphere,
the absorbance was recorded at 490 nm. A standard
toxicological parameter IC50 was calculated by non-
linear regression analysis of the inhibitory curves us-
ing GraphPad Prism software version 6 (GraphPad
Software, Inc., CA, USA).
Synthesis of N-phenylpyrazine-2-carboxamides
(1–25)
The corresponding pyrazinoic acid (5.0 mmol) was
dispersed in dry toluene (20 mL) and mixed with 1.5
eq. of thionyl chloride (0.55 mL, 7.5 mmol). The reac-
tion mixture was heated to reflux for approximately
1 h. Next, the excess of thionyl chloride was removed
by repeated evaporation with dry toluene under vac-
uum. The crude acyl chloride was dissolved in dry ace-
tone (20 mL) and added drop-wise to a stirred solution
of the corresponding aniline (5.0 mmol) with triethy-
lamine (5.0 mmol) in dry acetone (30 mL). The reac-
tion mixture was stirred at ambient temperature for
up to 6 h. The completion of the reaction was mon-
itored by TLC (eluent: hexane/ethyl acetate; ϕr =
2 : 1). The crude product adsorbed on silica gel by
solvent evaporation was purified by flash chromatog-
raphy (hexane/ethyl acetate gradient elution).
The analytical data of the prepared compounds
were fully consistent with the proposed structures and
are available in the Supplementary Data.
Anti-mycobacterial evaluation in vitro
Microdilution panel method (Servusova et al.,
2012). The anti-mycobacterial evaluation was car-
ried out by the Department of Clinical Microbiol-
ogy, University Hospital and Faculty of Medicine in
Hradec Králové, Charles University (Hradec Králové,
Czech Republic). Four mycobacterial strains were
used: M. tuberculosis H37Rv CNCTC My 331/88,
M. avium CNCTC My 80/72, M. avium CNCTC
My 152/73 and M. kansasii CNCTC My 235/80
(Czech National Collection of Type Cultures, Na-
tional Institute of Public Health, Prague, Czech Re-
public). The tested compounds were dissolved and se-
rially diluted in dimethylsulphoxide (DMSO), mixed
with Šula’s semi-synthetic medium (Trios, Czech Re-
public) and placed in a microdilution panel. The
tested species were added in the form of a suspen-
sion in an isotonic saline solution. The final conc−en1-
Results and discussion
Chemistry
Fig. 3 shows that the target derivatives (1–25)
were prepared by a convenient two-step synthesis us-
ing corresponding starting pyrazinoic acid, which was
treated with thionyl chloride to form carbonyl chlo-
ride. Various ring-substituted anilines were then used
for the aminolysis of carbonyl chloride to form final
compounds (1–25) (purified by flash column chro-
matography); see Table 1. The analytical data of all
the prepared compounds were in accordance with the
proposed structures and are available in the Sup-
plementary Data. The yields of chromatographically
pure products ranged from 17.8 % to 86.6 %. The
IR spectrum of all the compounds had a carbonyl
trations of tested compunds were of 100 g mL
50 g mL−1, 25 g mL−1, 12.5 g mL−1, 6.25 g mL−1
,
,
3.125 g mL−1 and 1.563 g mL−1), The final con-
centration of DMSO did not exceed 1 vol. %; this
concentration of DMSO did not affect the growth
of mycobacteria. The cultures were grown in Šula’s
semi-synthetic medium at pH 6.0 and 37◦C. The anti-
mycobacterial activity was determined visually after
14 days (6 days for M. kansasii and M. avium) of
incubation as the minimum inhibition concentration
(MIC, in g mL−1), i.e. the lowest concentration of
the tested substance that inhibited the growth of my-
cobacteria.
—
(C O) transmittance peak in the range of 1670–
—
1698 cm−1. The 1H NMR spectra exhibited amidic
hydrogen (-CONH-) as a broad singlet (independently
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J. Zitko et al./Chemical Papers
v
Fig. 3. Synthesis of final compounds (1–25); reagents and conditions: (i) SOCl2, toluene, reflux, 1 h; (ii) substituted aniline, TEA,
acetone, AT, up to 6 h.
Table 1. Summary of prepared compounds. Anti-mycobacterial activity against M. tuberculosis H37Rv and M. kansasii in vitro
expressed as MIC
Substituent
R2
MIC/( g mL−1
)
No.
Molecular
mass
logP
R1
R3
M. tuberculosis
M. kansasii
1
2
3
4
5
6
7
8
9
233.66
247.68
268.10
289.76
324.21
231.23
245.26
265.67
287.34
321.87
247.68
261.71
282.12
303.79
338.23
339.14
353.16
373.58
395.24
429.69
359.55
373.58
393.99
415.66
450.10
137.14
123.12
H
CH3
H
C(CH3)3
C(CH3)3
H
CH3
H
C(CH3)3
C(CH3)3
H
CH3
H
C(CH3)3
C(CH3)3
H
CH3
H
C(CH3)3
C(CH3)3
H
H
H
Cl
H
Cl
H
H
Cl
H
Cl
H
H
Cl
H
Cl
H
H
Cl
H
Cl
H
H
Cl
H
Cl
–
–
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
50
25
100
> 100
> 100
25
n.a.
> 100
3.13
> 100
12.5
3.13
50
> 50
> 100
> 100
> 100
25
1.15
1.85
2.05
3.28
4.18
1.23
1.94
2.13
3.36
4.26
1.63
2.34
2.53
3.76
4.66
2.43
3.14
3.33
4.56
5.46
2.50
3.21
3.41
4.63
5.53
–0.64
–1.31
2-CH3-5-F
2-CH3-5-F
2-CH3-5-F
2-CH3-5-F
2-CH3-5-F
3-Cl-4-CH3
3-Cl-4-CH3
3-Cl-4-CH3
3-Cl-4-CH3
3-Cl-4-CH3
2-CH3-5-I
2-CH3-5-I
2-CH3-5-I
2-CH3-5-I
2-CH3-5-I
2-I-4-Cl
2-I-4-Cl
2-I-4-Cl
2-I-4-Cl
2-I-4-Cl
–
n.a.
> 100
> 100
> 100
3.13
12.5
100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
3.13
> 50a
25
> 50a
> 50a
1.56–6.25
> 100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
INH
PZA
3.13
> 100
> 100
> 100
> 100
3.13
> 100
> 100
6.25
> 50a
> 100
> 50a
> 50a
0.2–0.78
12.5
CH3
H
C(CH3)3
C(CH3)3
–
–
–
a) Measurement at 100 g mL−1 not performed due to precipitation of tested compound in testing medium; INH – isoniazid, PZA
– pyrazinamide, n.a. – data not available.
of the solvent) in the range of δ 10.96–9.31. The
13C NMR spectra of all the compounds exhibited
carbonyl carbon (-CONH-) in the range of δ 168.2–
159.3.
itive effect on anti-mycobacterial activity within the
newly prepared series of compounds (1–25), the in-
dividual compounds were scored with points accord-
ing to the following rule: high activity, MIC = 3.13–
6.25 g mL−1, 3 points; moderate activity, MIC =
12.5–25 g mL−1, 2 points; weak activity, MIC = 50–
100 g mL−1, 1 point; no activity, MIC > 100 g mL−1
(MIC > 50 g mL−1 for compounds (22), (24) and
(25)), 0 points. Table 2 presents the scores of the
individual substituents for both M. tuberculosis and
M. kansasii activity; the number in parentheses is the
total number of active compounds (high, moderate or
weak activity) with the respective substitution. Fig. 4
graphically summarises the level of activity against
Anti-mycobacterial activity
Final compounds (1–25) were assayed in vitro for
whole cell anti-mycobacterial activity against M. tu-
berculosis H37Rv, M. kansasii, and M. avium using a
two-fold dilution microplate method. The results (Ta-
ble 1) are expressed as MIC ( g mL−1).
To determine the substituents in the pyrazine (R1,
R2) and anilide (R3) parts of the molecule with a pos-
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Table 2. Activity score of various substitution patterns and their hydrophobic substituent constants π
Anilide part (R3)
Pyrazine part (R1, R2)
5-CH3 6-Cl 5-tert-Bu 5-tert-Bu-6-Cl
2-Cl 2-CH3-5-F 3-Cl-4-CH3 2-CH3-5-I 2-I-4-Cl
H
0
π
0.56
M. tuberculosis 4 (3)
M. kansasii 1 (1)
0.64
5 (2)
2 (1)
1.04
8 (3)
6 (3)
1.84
3 (1)
0 (0)
1.91
3 (1)
5 (2)
0.7
0.9
2.13
3 (1)
0 (0)
3.03
0 (0)
0 (0)
8 (4) 5 (2) 7 (3)
9 (4) 2 (1) 3 (2)
π – Hydrophobic substituent constant; calculated as logP(RX) – logP(RH), where RX is substituted derivative and RH is non-
substituted derivative. See text for rules used for calculation of the score; numbers in parentheses represent the total number of
active compounds with the respective substitution.
Fig. 4. Level of antimycobacterial activity against M. tubercu-
losis H37Rv according to combinations of substituents
in pyrazine and phenyl part.
M. tuberculosis H37Rv according to combinations of
substituents R1, R2 and R3.
On reviewing the activity against M. tuberculosis
H37Rv as depicted in Table 2 and Fig. 4, it is obvious
that the most beneficial substitution on the phenyl
is R3 = 3-Cl-4-CH3, which produced two compounds
with high activity (12, 13) and one with moderate
activity (11), scoring 8 points. This substitution pat-
tern is fully compliant with the general formula GF
(Fig. 2), bearing the electron-withdrawing meta halo-
gen and electron-donating methyl in the para position.
Interestingly, this substitution pattern also performed
best against M. kansasii. The activity of compounds
with R3 = 2-CH3-5-F (6–10) and, in particular, R3 =
2-CH3-5-I (16–20) is probably reduced by the ortho
methyl group, the negative effect of which on anti-
mycobacterial activity was identified in the previously
published review (Dolezal et al., 2012). In the present
case, the negative effect of ortho methyl often coun-
teracts the positive effect of meta withdrawing halo-
gens. Similarly, ortho-halogen substitution was also
disadvantageous, e.g. R3 = 2-Cl in compounds (1–5)
and R3 = 2-I-4-CH3 in (22–25). Moreover, these com-
pounds lack the meta-withdrawing substituent, appar-
ently important for the anti-mycobacterial activity.
From the activities published in the review (Dolezal
et al., 2012), it appears that the phenyl part does not
tolerate ortho substituents (e.g. 2,6-Cl2, 2-CH3; 2-X-
5-OH, where X is halogen). This may be due to steric
hindrance with the structure of the potential target,
Fig. 5. Plot of anti-mycobacterial activity against M. tubercu-
losis H37Rv vs. calculated lipophilicity for compounds
(1–25);
– active, × – not active.
◦
although this cannot currently be confirmed.
Considering the pyrazine part of the molecule, the
best scores were achieved by non-substituted pyrazine,
followed by 6-Cl and 5-CH3 (it should be noted that
5-CH3 substitution might have been hampered due
to missing activity data for compound (7)). The
anilides derived from 5-tert-butyl-6-chloropyrazine-2-
carboxylic acid were completely inactive. This obser-
vation is in contrast with the SAR extracted from
the review (Dolezal et al., 2012), where the series de-
rived from 5-tert-butyl-6-chloropyrazine-2-carboxylic
acid possessed significant activities. However, these
compounds often bore the hydrophilic substituent
(R3 = 2-OH, 3-OH, or 4-OH) counteracting the
lipophilic substitution of the pyrazine part, or the
substituent with a small increase in lipophilicity such
as R3 = 3-F (πPh = + 0.16). The combination of
highly lipophilic phenyl substitution patterns used in
this study with a highly lipophilic substitution of the
pyrazine ring (R1 = tert-Bu, R2 = Cl) is, therefore,
disadvantageous.
Fig. 5 presents the plot of activity (log(1/MIC)
in M) against M. tuberculosis H37Rv vs. calculated
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J. Zitko et al./Chemical Papers
Table 3. Cytotoxicity of tested substances in HepG2 cells
vii
No.
IC50/ M
MIC of M. tuberculosis/ M
SI = IC50/MIC
9
11
12
13
18
21
> 50a (573.3)b
> 250a
> 100a (343.5)b
> 250a (820.7)b
> 50a (653.8)b
> 100a
10.9
50.5
12.0
11.1
8.4
> 4.59 (52.6)c
> 4.95
> 8.36 (28.7)c
> 22.54 (74.0)c
> 5.97 (78.0)c
> 5.75
17.4
a) Measurement at higher concentration not reproducible (precipitation of compound in cell culture medium); b) number in
parentheses represents hypothetic IC50 value calculated from the trend of inhibitory curve; c) number in parentheses represents SI
calculated based on hypothetic IC50 value.
lipophilicity logP. Among the active compounds, their
activity increased with lipophilicity. The compounds
with high activity (MIC = 3.13–6.25 g mL−1) pos-
sessed logP in the range from 2.34 to 3.36. This can
be regarded as the optimal lipophilicity in the series
under discussion. The compounds with logP ≥ 3.76
were inactive. This limit value can be deduced from
compounds (11–15) with the successful substitution
pattern R3 = 3-Cl-4-CH3, where compounds (11–13)
are active and compounds (14) (logP = 3.76) and (15)
(logP = 4.76) are completely inactive. However, it is
obvious that lipophilicity is only a secondary determi-
nant of anti-mycobacterial activity, probably facilitat-
ing the penetration through the lipophilic mycobacte-
rial cell wall. Compounds (4), (16), (17), and (22) lie
within the optimal lipophilicity range but still are in-
active. This can be explained by the negative influence
of substituent R3, for example, substituent (2-CH3, 2-
Cl, or 2-I) in the ortho position as discussed above or
non-compliance with the meta-EWG rule. This nega-
tive steric effect is obvious for 2-Cl and 2-I substitu-
tion; the effect of 2-CH3 is unclear, as compound (18)
(R1 = H, R2 = Cl, R3 = 2-CH3-5-I) is highly active,
but other compounds with the same phenyl substitu-
tion (16), (17), (19) and (20) are inactive.
Cytotoxicity
Drug-induced hepatotoxicity is an unfortunate
common side-effect of many of the first line anti-
tuberculosis agents (PZA, isoniazid, rifampicin) (Tost-
mann et al., 2008a). In view of the multidrug mix-
ture approach used for the treatment of tuberculo-
sis, it is highly probable that a newly developed anti-
tuberculosis compound would be used in combination
with clinically established agents. Due to possible tox-
icity synergism, the potential hepatotoxicity of new
compounds developed as potential anti-tuberculosis
agents should be assessed carefully. The human liver
carcinoma cell line (HepG2) is used as a model for hep-
atotoxicity studies in vitro (Singh et al., 2011; Tost-
mann et al., 2008b).
A hepatotoxicity assay of human HepG2 hepatoma
cells was performed in vitro for selected compounds:
see Table 3. The decrease in viability of the HepG2
cells was measured using a colorimetric assay (Kratky
et al., 2012; Owen, 1993) based on the reduction of
tetrazolium dye and the results were expressed as
IC50. The limited solubility in the culture medium of
all the compounds tested did not permit direct de-
termination of the exact IC50 value, but no toxic ef-
fect on HepG2 cells was observed up to the highest
concentration achieved during the measurement. The
IC50 values of compounds (9), (12–13), and (18) (Ta-
ble 3, values in parentheses) were calculated accord-
ing to the trend of the inhibitory curves by Graph-
Pad Prism software (version 6, non-linear regression
analysis). The inhibitory curves for compounds (11)
and (21) did not make it possible to calculate the
exact IC50 value. Nevertheless, the IC50 values of all
the compounds studied were hundreds of M, which
represents a significantly lower cytotoxicity than the
previously published anilides of 5-chloropyrazine-2-
carboxilic acid (IC50 mostly in units or tens of M)
(Zitko et al., 2013).
The most active compounds achieved MIC against
M. tuberculosis H37Rv (or M. kansasii) at the level
of 3.13 g mL−1 or 6.25 g mL−1, which are val-
ues comparable with the values of previously reported
anilides (of comparable MW) with MIC = 2–8
g
mL−1 (Dolezal et al., 2009) or MIC = 3.13–6.25
g mL−1 (Dolezal et al., 2011b). In recently pub-
lished data on anilides of 5-Cl-POA (Zitko et al.,
2013), the phenyl part tolerated a wider range of sub-
stituents than the anilides of non-substituted POA, 5-
CH3-POA, 6-Cl-POA, 5-tert-Bu-POA and 5-tert-Bu-
6-Cl-POA currently described or in the review pub-
lished previously. In the light of anti-mycobacterial
activity, 5-Cl-POA derivatives appear to be spe-
cial structures, as demonstrated by the ability of 5-
chloropyrazine-2-carboxamide to inhibit the mycobac-
terial fatty acid synthase I (Ngo et al., 2007; Sayahi
et al., 2012)
The estimated selectivity index (SI) defined as
IC50/MIC both in molar concentrations was calcu-
lated for activity against M. tuberculosis H37Rv. SI
values over 10 are regarded as safe for a drug can-
didate, granting sufficient difference between efficient
and cytotoxic concentrations.
None of compounds (1–25) exhibited any activity
against any of the two tested strains of M. avium.
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viii
J. Zitko et al./Chemical Papers
Conclusions
boxamides. European Journal of Medicinal Chemistry, 43,
1105–1113. DOI: 10.1016/j.ejmech.2007.07.013.
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Dolezal, M., Zitko, J., Osicka, Z., Kunes, J., Vejsova, M.,
Buchta, V., Dohnal, J., Jampilek, J., & Kralova, K. (2010).
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Twenty five new, previously undescribed anilides
of substituted pyrazinoic acids were synthesised by
known methods. The anti-mycobacterial activity of
the final compounds against M. tuberculosis H37Rv
and non-tuberculous mycobacterial strains of M. kan-
sasii and M. avium was evaluated in vitro. The
compounds so prepared supplement the large fam-
ily of anilides prepared by Doležal and co-workers.
It was shown that similar structure-activity relation-
ships pertain concerning the phenyl substitution pat-
tern. Three new anilides (12, 13, 18) of substituted
pyrazine-2-carboxylic acid exhibited whole cell anti-
mycobacterial activity in vitro against M. tbc H37Rv
with MIC around 10 M. Importantly, no cytotoxic-
ity in the HepG2 model was detected in vitro for the
tested concentrations and the estimated IC50 values
were in hundreds of M, indicating promising selec-
tivity. Compounds (11) and (21) exhibited significant
activity against M. kansasii with MIC = 3.13 g mL−1
(12.6 M and 8.7 M, respectively). No activity was
detected against M. avium.
Dolezal, M., & Kralova, K. (2011). Synthesis and evaluation of
pyrazine derivatives with herbicidal activity. In P. M. Larra-
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& Zitko, J. (2011). Antimy-
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Kos, J., Nevin, E., Soral, M., Kushkevych, I., Gonec, T., Bobal,
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Acknowledgements. This study was co-financed by the Eu-
ropean Social Fund and the state budget of the Czech Republic,
project no. CZ.1.07/2.3.00/20.0235, denoted as TEAB. This
study was also supported by the Ministry of Health of the Czech
Republic (IGA NZ 13346) and SVV 260 183.
Supplementary Data
The supplementary data associated with this arti-
cle can be found in the online version of this paper.
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